Dichotomy of the BSL phosphatase signaling spatially regulates MAPK components in stomatal fate determination

MAPK signaling modules play crucial roles in regulating numerous biological processes in all eukaryotic cells. How MAPK signaling specificity and strength are tightly controlled remains a major challenging question. In Arabidopsis stomatal development, the MAPKK Kinase YODA (YDA) functions at the cell periphery to inhibit stomatal production by activating MAPK 3 and 6 (MPK3/6) that directly phosphorylate stomatal fate-determining transcription factors for degradation in the nucleus. Recently, we demonstrated that BSL1, one of the four BSL protein phosphatases, localizes to the cell cortex to activate YDA, elevating MPK3/6 activity to suppress stomatal formation. Here, we showed that at the plasma membrane, all four members of BSL proteins contribute to the YDA activation. However, in the nucleus, specific BSL members (BSL2, BSL3, and BSU1) directly deactivate MPK6 to counteract the linear MAPK pathway, thereby promoting stomatal formation. Thus, the pivotal MAPK signaling in stomatal fate determination is spatially modulated by a signaling dichotomy of the BSL protein phosphatases in Arabidopsis, providing a prominent example of how MAPK activities are integrated and specified by signaling compartmentalization at the subcellular level.

Major points: 1. Although the proposed inhibitory roles of BSL2 and 3 in stomatal development is mostly clear, the same can't be said for BSU1. The conclusion that BSU1 can suppress stomatal development (e.g. Line 78) is mainly based on the bsl-q mutant, which, as the authors stated, contains T-DNA insertions at BSU1 and BSL1 and an amiRNA construct(s) against both BSL2 and 3. Compared with T-DNA mutants, the amiRNA approach has a potential drawback in its off-target effect, i.e., in this case, towards unknown BSL-related genes, and causes artifacts. Indeed, based on the authors' data of the double and triple T-DNA mutants, mutations in BSU1 have never resulted in higher stomatal lineage index, but rather led to significant drops, which contradict with the bsl-q phenotype. Thus, I suggest the authors to re-examine their data, and clarify the native roles of BSU1. Instead of relying solely on bsl-q, it would be ideal to assess the phenotype of T-DNA (or CRISPR)-based quadruple bsl1/2/3 bsu1 mutants. Examining a single bsl1 mutant with the same amiRNA against BSL2 and 3 (e.g. by crossing out the bsu1 in bsl-q) and comparing it with bsl-q may also help address the effect of both the amiRNA and BSU1. Though, I also think the authors have sufficient genetic data that a careful re-assessment of them may suffice. Further, I found the section describing the double/triple mutant phenotypes somewhat confusing, and improvements on its presentation would be needed.
2. The in vitro assay that assessed the effect of BSL2 on MPK6 activity (Fig. 6a) used the phosphorylation status of MBP as a readout of MPK6 activity. However, the observed effect on MBP could be due to the BSL2 activity acting on the MBP protein itself. Thus, probing the effect on phosphorylated MPK6 (by anit-p42/44 or phos-tag gel) in the presence of BSL2 would be a superior readout and will better support the authors' claim that BSL2 acts on MPK6 directly. Further, the amount of the 5x BSL2 reaction seems off in the assay as judged by the anti-GST blot.
3. The manuscript would be strengthened by having some initial investigations on whether the membrane/nuclear distribution of BSL2/3/BSU1 is dynamic or not. Given that BSL2/3 can interact with BASL, quantification of their distribution in stomatal lineage cells at different developmental stages, similar to what the authors have showed for BSL1 in their previous work, would be a reasonable start.
Other points: -What is the transcript level of BSL2 and 3 in the bsl-q mutant compared with WT? -A few genetic materials and data (e.g. TMMp:BSL1-mRFP and localization of BSL2/3 and BUS1) were first appeared in the authors' last publication on BSL1. The authors should refer to the work when first presenting them to distinguish the novelties of the current study.
-The loading control (rubisco staining) in Fig. 6b is not clear. Since quantitative data were presented, it'd be better to represent loading by probing control proteins by Western blot.
Reviewer #2 (Remarks to the Author): Stomata formation is regulated by a cascade of MAP kinases. The manuscript of Guo and Dong describes regulation of this cascade by four BSL phosphatases. Previously, this group showed that one of these phosphatases, BSL1, activates MAPKKK YODA at the plasma membrane. Here, they show that at the plasma membrane all four phosphatases dephosphorylate YODA which activates it. But in the nucleus, three out of the four phosphatases dephosphorylate a MAPK MPK6 which inactivates it. The authors clearly demonstrate that when subcellular localization of these phosphatases changes, their substrate changes too. This is an elegant paper that significantly contributes to our understanding of the regulatory mechanism of MAPK cascades, and it demonstrates how a phosphatase can function as both a positive and negative regulator of a signaling pathway. In addition, it provides a great example of how the nature of an enzyme's substrates can depend on the enzyme's subcellular localization. The data are comprehensive, of high quality and support the conclusions. The writing is clear and logical. Minor suggestions: 1. Figure 1B. Images (d) and (e) show different phenotypes. There is much more stomata clustering in (e) compared to (d). However, on the graph, the phenotype of these two mutants has no statistical difference. Are these images representative? Is SLI a good measurement of these phenotypes? 2. The use of asterisks in Fig 1B is confusing. A compact letter display would be better for visualization in this figure.

Reviewer #1 (Remarks to the Author):
In this manuscript from the Dong lab, the authors explored the function of the BSL2, BSL3 and BSU1 phosphatases in stomatal development. The authors previously showed that BSL1, a member of the BSL family, is associated with the polarity complex on the cell membrane and activates the MAPKKK YODA in suppressing stomatal development. Here, using a combination of genetic, microscopic and biochemical approaches, they propose that the other three members of the family can function in the nucleus, and, in contrast to the role of BSL1, promote stomatal development by inhibiting nuclear MPK6.
The finding that the BSL phosphatases exhibit opposing functions in a cell compartmentdependent manner is novel and intriguing, and the authors presented a mechanism for the inhibitory effect in the nucleus. The work also helps dissect the role of individual BSLs in stomatal development. The experiments were generally well designed and executed. There are, however, a few points that I'd like the authors to address.

Response:
We thank the reviewer's appreciation of our work.
Major points:1. Although the proposed inhibitory roles of BSL2 and 3 in stomatal development is mostly clear, the same can't be said for BSU1. The conclusion that BSU1 can suppress stomatal development (e.g. Line 78) is mainly based on the bsl-q mutant, which, as the authors stated, contains T-DNA insertions at BSU1 and BSL1 and an amiRNA construct(s) against both BSL2 and 3. Compared with T-DNA mutants, the amiRNA approach has a potential drawback in its off-target effect, i.e., in this case, towards unknown BSL-related genes, and causes artifacts. Indeed, based on the authors' data of the double and triple T-DNA mutants, mutations in BSU1 have never resulted in higher stomatal lineage index, but rather led to significant drops, which contradict with the bsl-q phenotype.

Response:
We thank the reviewer for sharing the insights about interpreting the mutant phenotypes. We are also thankful for the constructive suggestions that will greatly improve our data quality.
Here is what we understand about the quadruple mutant phenotypes. We do believe BSU1 contributes to the collective role of BSL1/BSL2/BSL3 at the PM to suppress stomatal formation, besides its positive role in the nucleus. This conclusion was based on the observations explained below. 1) Probably due to the mutagenesis feature (T-DNA combined with amiRNA), this quadruple mutant displays phenotypic severity at varying levels and shows a uniquely striking phenotype of large patches of stomatal guard cells, which often appear at the leaf edges (see below, blue arrows) but never observed in the triple mutant bsl1;bsl2;bsl3. On the other hand, when SLI (stomatal lineage index) is calculated, we have been conventionally using similarly central regions of the cotyledons to collect the numbers (red box below), where the large patches in the quadruple mutant were often missed for counting. This explains why ultimately the quantification of stomatal lineage index in the quadruple mutant was shown slightly higher than that of the triple mutant of bsl1;bsl2;bsl3 (Fig. 1). We added more explanation in the main text to inform the mutant phenotype (see screen capture below).
2) The other supporting evidence of BSU1, like BSL1/BSL2/BSL3, contributing to the negative regulation of stomatal formation at the PM came from the overexpression data of BSU1 vs. myr-BSU1. Indeed, the less nuclear partition of BSU1 (myr-BSU1) induced less severe stomatal overproduction versus the predominant nuclear BSU1 that induced severe stomatal overproduction ( Fig. 2 and 3). The results suggested more BSU1 partition at the PM is associated with a negative regulation of BSU1 in stomatal formation.
3) To explain why bsu1 mutation reduces stomatal production of the single or double mutations among bsl1, bsl2, or bsl3 ( Supplementary Fig. 2) but instead increased that of the triple mutant bsl1;bsl2;bsl3, we believe one should consider the signaling pathway of tiered YDA-MKK4/5-MPK3/6 cascade, so that the seemingly contradictory phenotype can be reconciled by the fact of YDA being an upstream activator of MPK3/6. According to our model, the activation of upstream YDA is triggered by all four members (PM function), whereas the inhibition of MPK3/6 is controlled by BSL2/BSL3/BSU1 (nuclear function). In the quadruple mutant, the absence of four BSL members leads to the loss of YDA activity at the cell cortex, resulting in the loss of MPK3/6 activity in the nucleus. Therefore, in bsl-quad, regardless of the removal of BSL2/BSL3/BSU1mediated inhibition on MPK3/6 in the nucleus, because of no YDA activating MPK3/6, the overall phenotype in the quadruple mutant still shows the YDA loss of activity, thereby the enhanced overproduction of stomata in bsl-q. On the other hand, in the triple mutant bsl2;bsl3;bsu1, in which BSL1 is present and YDA is active, the removal of BSL2/BSL3/BSU1-mediated inhibition of MPK3/6 would lead to an enhanced MPK3/6 activity, resulting in suppressed stomatal production (as we see Fig. 1 and Supp. Fig. 2).
Thus, I suggest the authors to re-examine their data, and clarify the native roles of BSU1. Instead of relying solely on bsl-q, it would be ideal to assess the phenotype of T-DNA (or CRISPR)-based quadruple bsl1/2/3 bsu1 mutants. Examining a single bsl1 mutant with the same amiRNA against BSL2 and 3 (e.g. by crossing out the bsu1 in bsl-q) and comparing it with bsl-q may also help address the effect of both the amiRNA and BSU1. Though, I also think the authors have sufficient genetic data that a careful re-assessment of them may suffice Response: We attempted to search for a null bsl-quadruple mutant from a crossed population (F2) of bsl1;bsu1 X bsl2;bsl3, in which we identified all the mutation combinations in this study, but realized that the mission is impossible at this stage, because BSU1 and BSL2 are two closely linked genes (see below) and bsl2 bsl3 double is sterile. Although we have germinated seeds to cross mutants bsl1;bsl2;bsu1 with bsl3 for the revision, we are not able to identify a true null quadruple mutant in a relatively short time window.
Indeed, the reviewer's suggestion of re-examination of the amiRNA lines is highly valuable. To rule out the possibility of off-target effect of amiRNA construct, as advised, we took strategies below.
1) First, we used qPCR to evaluate the transcript levels of BSL1 and BSU1 in the amiRNA-BSL2/3 containing plants, i.e. amiRNA-BSL2;3, bsl1 amiRNA-BSL2;3, and bsu1 bsl1 amiRNA-BSL2;3 (see data below). The results show that similar expression levels of BSL1/BSU1 were observed in wild type vs. the silencing line amiRNA-BSL2;3, whereas their expression levels were almost under detection when the corresponding T-DNA insertional mutation was present. The results clearly demonstrated that no off-target effects of amiR-BSL2;3 were observed on the homologous family members, BSL1/BSU1.
3) Accordingly, we made major textual changes by adding a whole section to clarify the contribution of BSU1 (see screen captures below).
Further, I found the section describing the double/triple mutant phenotypes somewhat confusing, and improvements on its presentation would be needed.

Response:
We are thankful for the suggestion. After we added more details about characterization of the BSU1 contribution, the reading flows much better and helps the transition to the double and triple mutants. The edits were tracked below.
2. The in vitro assay that assessed the effect of BSL2 on MPK6 activity (Fig. 6a) used the phosphorylation status of MBP as a readout of MPK6 activity. However, the observed effect on MBP could be due to the BSL2 activity acting on the MBP protein itself. Thus, probing the effect on phosphorylated MPK6 (by anit-p42/44 or phos-tag gel) in the presence of BSL2 would be a superior readout and will better support the authors' claim that BSL2 acts on MPK6 directly. Further, the amount of the 5x BSL2 reaction seems off in the assay as judged by the anti-GST blot. Response: We thank the reviewer for pointing out this possibility. As suggested, we performed in vitro phosphorylation assays this time using MPK6 as substrate. The phosphorylation status of MPK6 was detected by anti-p42/44 antibody. Similar to what we found with the MBP substrate, we identified lowed MPK6 phosphorylation levels when more BSL2 is added in the system (data shown below). This time, the amount of BSL2 was better adjusted and shown by anti-GST blots.
3. The manuscript would be strengthened by having some initial investigations on whether the membrane/nuclear distribution of BSL2/3/BSU1 is dynamic or not. Given that BSL2/3 can interact with BASL, quantification of their distribution in stomatal lineage cells at different developmental stages, similar to what the authors have showed for BSL1 in their previous work, would be a reasonable start. Response: We thank the reviewer for sharing deep thoughts. As advised, we added the data of dynamic distribution of BSL2-mRFP when co-expressed with the polarity marker GFP-BASL. Results indeed showed the subcellular distribution of BSL2 can be dynamically changed at different stages in stomatal development (see data and textual additions below).
Other points: -What is the transcript level of BSL2 and 3 in the bsl-q mutant compared with WT? Response: As requested, we checked the transcript level of BSL2 and 3 in the wild type and bsl-q mutant. The data is included part of Supplementary Fig. 1b.
-A few genetic materials and data (e.g. TMMp:BSL1-mRFP and localization of BSL2/3 and BUS1) were first appeared in the authors' last publication on BSL1. The authors should refer to the work when first presenting them to distinguish the novelties of the current study. Response: We thank the reviewer for careful reading of the manuscript. The references have now been included when the published materials were first appeared in this manuscript.
-The loading control (rubisco staining) in Fig. 6b is not clear. Since quantitative data were presented, it'd be better to represent loading by probing control proteins by Western blot. Response: As requested, the experiment was repeated to give improved results (see below). The Ponceau-S staining of Rubisco was replaced with Western Blotting using anti-MPK6 to show MPK6 protein levels in vivo.